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Even though a detailed understanding of the proliferative characteristics of T lymphocytes is imperative in many research fields, prior studies have never reached a consensus on these characteristics, and on the corresponding cell cycle kinetics specifically. In this study, the general proliferative response of human T lymphocytes to phytohaemagglutinin (PHA) stimulation was characterized using a carboxyfluorescein succinimidyl ester-based flow cytometric assay. We were able to determine when PHA-stimulated T lymphocytes complete their first division, the proportion of cells that initiate proliferation, the subsequent division rate of the cells, and the impact of irradiation on these proliferative properties. Next, we accurately visualized the cell cycle progression of dividing T lymphocytes cultured in whole blood using an adapted 5-ethynyl-2’-deoxyuridine pulse-chase method. Furthermore, through multiple downstream analysis methods, we were able to make an estimation of the corresponding cell cycle kinetics. We also visualized the impact of X-rays on the progression of the cells through the cell cycle. Our results showed dose-dependent G2 arrest after exposure to irradiation, and a corresponding delay in G1 phase-entry of the cells. In conclusion, utilizing various flow cytometric assays, we provided valuable information on T lymphocyte proliferation characteristics starting from first division to fully dividing cells.

Background: Both standardized and non-standardized candida skin tests are used in clinical practice for functional in-vivo assessment of cellular immunity with variable results and are considered not reliable under the age of 1 year. We sought to investigate the reliability of using manually prepared candida intradermal test in the evaluation of T cell function in infants during their second year of life. Methods: Twenty-five healthy infants were tested with manually prepared intradermal candida test. Cultured lymphocytes were stimulated with phytohemagglutinin (PHA) and gamma interferon (IFN-γ) levels were measured in the culture supernatant of stimulated and non-stimulated samples using ELISA. Results: The enrolled infants were 14 to 24 months old (mean 19.2 ± 3.13 months). They were 17 boys (68 %) and 8 girls (32 %). Candida skin test was positive in 17 out of the 25 infants (68%). All infants showed increased IFN-γ levels after PHA stimulation (mean ± SD: 0.83±0.29 ng/ml) compared to basal levels (mean ± SD = 0.16 ± 0.16 ng/ml). The increase of IFN γ levels after PHA stimulation ranged from 1.54 to 38 folds. Infants with positive and negative candida tests showed comparable results in terms of clinical and immunological assessment except for weight percentiles for age that were higher among candida positive group. Conclusion: Candida intradermal test is a cost-effective simple test for evaluation of T cell function with 70 % sensitivity in healthy infants above the age of one year. Keywords: Candida, IFN-γ, infants, intradermal, lymphocyte proliferation, PHA stimulation, T cell, Tuberculin

article i nfo Lymphocytes proliferation after antigen-driven activation leads to an increase in cell count, which could last some week, until apoptosis mechanisms allow the homeostatic control of the system. During the first days of this stimulation, activated lymphocytes display high resistance to apoptosis and to most immunosuppressive drugs. According to the literature, few compounds have been described to kill recently activated cells, by inhibiting metabolic processes fundamental to proliferation. The aim of our work was to evaluate comparatively these different compounds, in order to identify the best strategy to kill cells that have undergone proliferation, while sparing the repertoire of resting cells. After preliminary experiments, 3-HAA and bortezomib were selected as the most suitable compounds for our purposes. The possible synergic effect of 3-HAA with bortezomib or with manganese ions was also assessed. 3-HAA was confirmed to be the most reliable pharmacologic approach to inhibit proliferation with acceptable toxicity on resting cells. While in the case of PHA stimulation 3-HAA led to death of most lymphocytes, only a minor percentage of cells were killed after allo-stimulation, suggesting that the effect is proportional to the percentage of stimulated lymphocytes. Manganese ions further enhanced this effect, while results with bor- tezomib seemed to be less consistent. These results deserve further investigations to develop new procedures for targeting activated cells with pharmacological approaches.

A simple method of freezing lymphocytes requiring a cryoprotective medium consisting of glucose, MEM, and dimethylsulfoxide (DMSO) and an ultra-low freezer to freeze and store the lymphocytes is described. The thawed lymphocytes following 2 weeks' to 5 months' storage were found to be useful for HL—A antibody screening, HL—A typing, and PHA stimulation.

Steady-state sickle cell disease (SCD) patients may have increased plasma levels of acute phase reactants and pro-inflammatory cytokines because of subclinical inflammation. We have estimated TNF-alpha levels in the plasma and in supernatants following peripheral blood mononuclear cell (PBMC) activation with phytohemagglutinin (PHA) in a group of Kuwaiti SCD patients using ELISA. The group consisted of 28 SS, 8 Sbeta-thal, and 2 SD patients all in steady state; 5 SS patients were studied during 7 episodes of painful crisis. The subjects were aged 2 to 16 years, with a mean of 7.3 +/- 3.5 years. The beta(S)-globin gene cluster haplotype, alpha-tha1 status, and spleen function were determined in the SS group using standard techniques. Most (82%) were homozygous for the Saudi Arabia/India haplotype and had elevated Hb F levels ranging from 15% to 35%. There were 24 controls (Hb AA or AS), of whom 14 were healthy and 10 were acutely ill at the time of the study. None of the children with SCD (either in steady state or crisis) had detectable plasma TNF-alpha, but four controls (3 acutely ill and one healthy) had levels ranging from 61.7 to 249.8 pg/mL. Following PHA stimulation most subjects responded with high levels of TNF-alpha, with the median level among the steady-state SS patients being significantly higher than that in the controls (both the acutely ill or healthy). It therefore appears that because of the mild disease among our Arab SS children, TNF-alpha is not detectable in their plasma in steady state; these children, however, had a significantly higher response than controls following PBMC activation.

A national survey of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) in emission gases from the municipal waste incinerators in 1997 revealed that the Nose Bika Center was heavily contaminated by PCDF. Ninety-four workers underwent a physical examination, and blood biochemistry, lymphocyte marker, and NK activity studies were carried out, along with blood dioxin measurements. Information on working history, life-style, and dietary habits was obtained by questionnaire and interview. The blood dioxin levels were as follows. The median TEQ of dioxins was 39.7 pg I-TEQ/g lipid, and the range was 13.3 to 805.8. The median 2,3,7,8-TCDD concentration was 3.9 pg TEQ/g lipid, and the range was

When endothelial cells (EC) were stimulated with IL-1 and/or lymphocytes with rIL-2 or PHA, the binding of lymphocytes to EC was increased. PMA treatment of lymphocytes alone did not increase their binding to EC, but when EC were additionally induced with IL-1 the binding was increased. The expression of LFA-1 was constant, whilst the expression of CD49d was increased after rIL-2 and PHA stimulation. The PMA- and rIL-2-induced lymphocyte binding to IL-1-induced EC was inhibited by anti-CD11a, CD18 and CD49d mAbs; on the other hand, the enhanced binding of PHA-stimulated lymphocytes to EC could not be blocked by these mAbs. These results show that activation of lymphocytes by various stimuli leads to different usage of adhesion pathways in their binding to inflammatory EC.

We studied the toxic and genotoxic effects of UV light and an alkylating agent (methyl methanesulphonate; MMS) in human peripheral blood lymphocytes (PBL) in Go and after PHA stimulation. We measured DNA repair synthesis and replication in treated PBL together with deoxynucleoside triphosphate pool and ATP concentrations. In addition to its genotoxic action, MMS induced a strong toxic effect in cycling cells, as shown by a marked depletion of nucleotide pools.

The purpose of this study was to determine the effect of T-lymphocytes and phytohemagglutinin (PHA), a T-cell mitogen, on the expression of bovine leukemia virus (BLV) in cultured B-lymphocytes from BLV-infected cattle and sheep. Bovine B-lymphocytes were isolated by negative selection via complement-mediated lysis of T-lymphocytes. Additionally, bovine and ovine B-lymphocytes were positively selected using fluorescence activated cell sorting. Expression of BLV in cultured bovine and ovine B-lymphocytes occurred in the absence of T-lymphocytes and without PHA stimulation. The results of this study demonstrate that BLV replication in cultured B-lymphocytes is T-cell independent. This finding may have implications for the mechanism of viral latency within infected B-lymphocytes.

X-ray irradiation on blood products is a common method to prevent the risk of inducing PTGVHD, and has recently become to be used in Japan. We have tried X-ray irradiation on WB, CRC and PC using HITACHI X-ray Irradiation Apparatus MBR1520R, and studied its practical usefulness.In case of irradiation on blood bags as the minimum dose of 1, 500 rads, these conditions are thought to be practical: 1) the use of 1.0mm Al filter 2) the distance of 550nm from X-ray source 3) irradiation on 4 bags at the same time. But, it has also been noticed that total doses and qualities of X-ray absorbed into blood were different between upper and lower side of the bag. Bloods on upper side absorbed much doses and a wide range of X-ray, on the other hand, bloods on lower side absorbed less doses and hard X-ray.In these conditions, irradiated lymphocytes showed a complete inhibition of thymidine uptake in MLC test, still had 15% of activity in PHA stimulation. The qualities of other blood components have not changed before and after irradition.X-ray irradiation is useful in a routine work of blood center, but problems of proper doses and a uniformity of irradiation are remained to be sloved.

Ageing appears to affect immune and neuroendocirne function in horses and response to acute exercise. No studies have examined the combined effects of training and ageing on immune and neuroendocirne function in horses.To ascertain whether training and age would affect the plasma beta-endorphin (BE) and cortisol (C) as well as immune function responses to acute exercise in Standardbred mares.Graded exercise tests (GXT) and simulated race tests (SRT) were performed before and after 12 weeks training at 60 % HRmax. BE and C were measured at rest and at 5, 10, 20, 40, 60 and 120 min post GXT. Leucocyte cell number, CD4+ and CD8+ lymphocyte subsets, and mitogen stimulated lymphoproliferative response (LPR), were measured in jugular blood before and after the SRTs.Cortisol rose by 5 min post GXT in young (Y) and middle-age (MA) mares (P0.05) and remained elevated until 40 and 60 min post GXT, respectively during both pre- and post training GXT. There was no rise in C in old (0) mares after either GXT (P0.05). Pretraining BE rose (P0.05) by 5 min post GXT in all mares. After training, BE was higher in Y and O vs. MA (P0.05) at 5 min post GXT. Post training BE was higher at 5 min post GXT in Y and O vs. pretraining (P0.05). After SRT, lymphocyte number rose in all mares (P0.05); however, lower lymphocyte numbers (P0.05) were seen in MA vs. Y and O vs. MA (P0.05). The O had reduced LPR to Con A and PHA stimulation (P0.05) compared to Y and MA after the SRT after both pre- and post training SRT. LPR to PWM was lower (P0.05) in O vs. Y and MA after the pretraining SRT. Training caused an increase in resting LPR to PWM in MA only (P0.05).Both age and training altered the plasma beta-endorphin and cortisol responses as well as and immune responses to acute exercise.This study provides important information on the effects of ageing and training that will aid in the management and care of an increasing number of active older horses.

Progesterone is an endogenous immunomodulator and can suppress T-cell activation during pregnancy. We have previously shown that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non-genomic inhibition of Na+/H+-exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)-induced T-cell proliferation. The presence of amiloride-sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20α-hydroxyprogesterone (20α-OHP). Furthermore, 20α-OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone-BSA demonstrated non-genomic action via plasma membrane sites. Finally, co-stimulation with PHA and progesterone or amiloride, (5-(N, N-dimethyl)-amiloride, DMA), inhibited PHA-induced T-cell proliferation, but this inhibition did not occur with 20α-OHP and PHA co-stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA-induced T-cell proliferation. This is the first study to show that progesterone causes a rapid non-genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20α-OHP. The inhibition of NHE1 leads to immunosuppressive T-cell proliferation and suggests that progesterone might exert a major rapid non-genomic suppressive effect on NHE1 activity at the maternal–fetal interface in vivo and that 20α-OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface. J. Cell. Physiol. 211: 544–550, 2007. © 2007 Wiley-Liss, Inc.

Present study has shown that a total cytokine pool produced by human blood cells in presence of proteins from the γ-globulin fraction, or their complexes with copper and zinc, as well as with pure copper and zinc cations, contains up to 148.0±14.9 pg interleukin-1β (IL-1β) per mL. The γ-globulins modified by copper or zinc binding induce, resp., 2.0 and 1.4-fold less IL-1β than intact proteins (significant by p = 0.1). Meanwhile, the copper-γ-globulin proved to be 2.7-fold more active (р < 0.01) IL-1β inducer, as a zinc-protein complex. Pure zinc ions did not induce IL-1β production, whereas induction rates with pure copper ions are 1.6-fold higher (p < 0.05) than with γ-globulin, and 1.45-fold more efficient that PHA stimulation (p < 0.1). A potential participation of copper and zinc cations bound to antibodies from the microenvironment and, thus, transforming Fc regions of antibody molecules, is discussed with regard of regulation of IL-1β production by human blood cells induced by activation of their Fc receptors.

Research conducted at McMaster University has shown a relative biological effect (RBE) of unity, between 280keV neutrons and 137 Cs gamma radiation, for the induction of apoptosis in human lymphocytes. Similar results have also been seen by Vral et al. (1998) and Warenius and Down (1995) for the induction of apoptosis in human lymphocytes and mouse thymocytes respectively. It has previously been reported that neutrons of energies 36keV to 14.6Mev have an RBE ranging from 67.1±28.9 to 16±6.8 for induction of dicentrics in human lymphocytes respectively (Schmid et al., 2003, 2002, and 2000). This discrepancy between RBEs suggests that further work is needed to understand why human lymphocytes have such varying response to different radiations. Radiation‐induced apoptosis and chromosomal aberrations are being measured in parallel. In this study, it was found that 280keV neutron induce chromosome breaks with an RBE of 6.4 (for the induction of 1 break/cell). An experiment was established to study the types of chromosomal aberrations which exist in the lymphocytes which have not committed apoptosis 48 hours after radiation. Isolated lymphocytes were incubated for 48 hours in complete media before PHA stimulation. This allowed the analysis of the chromosomal damage present in cells that do not commit apoptosis immediately. It is possible that the cells which have not committed apoptosis at 48 hours are those which have been less severely damaged and may be viewed by the organism as being repairable.

Experiments were carried out using human lymphocytes in order to test the effect of pH shifts on radiosensitivity of cells irradiated in the G 2 stage. In our culture conditions constant variations in medium pH over the range of 7.4–6.8 were observed as a function of incubation time after PHA stimulation; in addition the pH of the medium was adjusted in parallel cultures over the range of 5.9–8.2. Then we exposed the cultures with different pH to a treatment of X-rays delivered 2 h before fixation. The pH of the medium was found to greatly affect the yield of induced chromatid aberrations.

The effects of bilateral olfactory bulb (OB) ablation on pressor and behavioral responses to stimulation of the posterior hypothalamic area (PHA) and midbrain reticular formation (MRF) were investigated in unanesthetized and unrestrained rats with chronic electrodes and arterial cannula implants. Bilateral olfactory bulbectomy induced a marked increase in emotional responses to given stimuli. A high incidence of muricide was also observed after olfactory bulbectomy. The threshold for inducing behavioral arousal and pressor response to PHA and MRF stimulation significantly decreased at 5 and 10 days after olfactory bulbectomy. The pressor responses to either PHA or MRF stimulation were markedly enhanced. The enhancement in pressor response to MRF stimulation occurred immediately after olfactory bulbectomy and persisted throughout the experiment, while that to PHA stimulation appeared gradually and reached maximum at 10 days after olfactory bulbectomy. These results suggested that olfactory bulbectomy has a great influence not only on emotionality but also on the central neural mechanisms of autonomic regulation.

Spontaneous SCE frequencies were measured in cells undergoing their second mitosis at 54, 68, 72, 78 or 90 h after PHA stimulation of whole blood cultures from 7 normal donors. A consistent pattern of fluctuation of SCE levels was observed between 54 and 78 h. The magnitude of change in SCE frequency between consecutive fixation times was as high as 80% for some donors. These observations support the hypothesis of Snope and Rary (1979) that there are subpopulations of lymphocytes with different spontaneous SCE levels and different proliferation rates. In untreated cancer patients the patterns of change in SCE frequency with time were different from that seen in normal donors. This may be because of quantitative changes in T-lymphocyte sub-populations which have been observed in cancer patients. Changes in SCE frequency of less than about a factor of two, observed at a single sampling time may not be indicative of genotoxic events or genetic instability but simply represent changes in the composition of lymphocyte sub-populations and/or in their rates of proliferation in vitro.

We have tested G0 phase human peripheral lymphocytes for liquid holding recovery (LHR) mediated decreases in X-ray-induced chromosomal aberration yields and increases in sister-chromatid exchange (SCE) levels such as have been demonstrated for confluency-inhibited mouse cells in culture (Nagasawa and Little, 1981). No influence on either aberration yields or SCE levels was demonstrated. However, an effect at least superficially similar to the LHR effect was seen for rings and dicentrics, but not for deletions or SCE in lymphocytes in transition between G0 and G1 following PHA stimulation.

Four Rhesus monkeys were subjected to daily administration of 2.5 mg of tetrahydrocannabinol (THC) /kg body wt, after establishing the norms for complete blood count, T- and B-cell concentrations, and the dose response of thymidine incorporation after PHA stimulation. THC was administered daily for 3 weeks, the treatment was stopped, and then the animals were allowed to recover for 4 weeks. Cellular responses, incorporation studies and fibrinogen levels were determined during the treatment and recovery phases. Compared to 4 vehicle-treated animals, the THC-treated animals experienced significant augmentation of both their total white cell and their neutrophil counts during the treatment phase which returned to normal levels during the recovery phase. There was no alteration in total lymphocyte count or T- or B-cell concentrations. Fibrinogen levels of the THC-treated animals during the treatment phase were also elevated compared to controls, and the levels diminished to the same values as the vehicle-treated animals during the recovery phase. Possible mechanisms for the response of Rhesus monkeys to short-term administration of THC are discussed.

Peripheral blood lymphocytes of patients with chronic polymorphic light eruption (n = 33) show a normal blastogenic transformation after PHA stimulation. The excision repair mechanism is unaltered. There is no evident difference between lymphocytes taken during light eruption and those taken in an interval. A correlation to the level of IgE is not demonstrable.

Vor und wahrend einer therapeutischen Bestrahlung mit 60Co wurden die PHA-Stimulierbarkeit und das cytochemische Verhalten der Lymphocyten bei Patienten mit M. Hodgkin sowie die PHA-Stimulierbarkeit der Lynphocyten bei Patienten mit Bronchialcarcinom untersucht. Unter der Bestrahlung kam es bei den Patienten mit M. Hodgkin, bei denen eine erste supradiaphragmale Bestrahlungsserie durchgefuhrt wurde, und bei den Patienten mit Bronchialcarcinom zu einer signifikanten Abnahme der PHA-Stimulierbarkeit der Lymphocyten. Nicht stimulierte Lymphocyten-Kontrollkulturen wiesen unter der Bestrahlung keine Anderung des 3H-Thymidineinbaus auf. Die cytochemisehen Untersuchungen zeigten bei den Patienten mit M. Hodgkin unter der Bestrahlung einen Anstieg des PAS-Index der Lymphocyten sowie einen Anstieg des Beta-Glucuronidase-Gesamtindex bedingt durch eine Zunahme des Index der Reaktionstyp-B-Lymphocyten. Der feulgenphotometrisch bestimmte DNS-Gehalt der Lymphocyten anderte sich unter der Bestrahlung nicht.

The growth in volume of human peripheral blood lymphocytes after stimulation with various concentrations of PHA was measured with an electronic particle counter. The percentage of growing cells and averaged values describing their growth rates during the elapsed period of culture were estimated by fitting to the observed data the volume distributions derived from a mathematical model. With sub-optimal doses, the percentage of cells stimulated , and their incremental growth rate, increased with increasing dose of PHA, but the time-course of recruitment into the G1-phase was similar with all PHA concentrations studied. The results provide strong support for the 'trigger' hypothesis that there is a distribution of stimulation thresholds within the lymphocyte population: consequently, increasing mitogen concentration will be expected to result in increased numbers of responding cells within the suboptimal concentration range.

Human peripheral lymphocytes were stimulated with phytohemagglutinin (PHA) in liquid culture, suspended in agar and incubated in glass capillary tubes. Compact colonies of lymphocytes were found growing along the tube bottom in a buffer film, while compact clusters and rare diffuse colonies were observed inside the agar. Several parameters affecting the clonal growth were studied and optimized: PHA-dose, agar concentration, gel length (volume), quantity and density of seeded cells per capillary and gel length. Colony yield mainly depends on the seeded-cell density with a sharp optimum at 2 × 105 cells/ml irrespective of gel length; higher cell densities reduce the colony yield, suggesting that colony growth is the result of both stimulatory and inhibitory factors produced by cooperating cells. Following the daily clonal growth was only possible with undisturbed tubes; the number of colonies steadily increased from day 2 until day 7. Densitometric colony scanning is possible, yet problematic. Colony yield (plating efficiency) in 10–50-fold higher in agar capillaries than in the usual Petri dishes. An additional advantage is that the capillaries provide a basis for a simple and reliable assay system for determining regulatory factors of lymphocyte proliferation (including chalones).

Cord blood analysis of 21 infants indicated a deficient immunological status of newborn babies as revealed by the content of T lymphocytes and bactericidal activity of leucocytes; however,3H thymidine uptake of lymphocytes following PHA stimulation was normal. Serum IgG levels were high at birth as compared with IgM and IgA. The hemoglobin, total protein and albumin levels in plasma were mostly within normal limits.

SUMMARYThe rare disorder of chronic mucocutaneous candidiasis is described in a two-year-old African girl who had a moderate lymphopenia due to fewer T and B cells, impaired lymphocyte transformation to PHA and candida antigen, decreased leucocyte migration inhibition to candida antigen and a defective cutaneous delayed hypersensitivity reaction to candida antigen and dinitrochlorobenzene. The patient's serum had a marginal inhibitory effect on the transformation of normal lymphocytes to candida antigen and no effect on PHA stimulation of these cells, as measured by stimulation index. In addition, IgA was deficient in her serum. No associated endocrinopathy was detected and she had a mild iron deficiency anaemia. There was a rapid and excellent response to ketoconazole with all lesions clearing within two months of treatment.

The influence of protein depletion on serum factors in PHA lymphocyte blastogenesis was studied in a rat model. Buffalo rats were divided randomly into two groups and fed either a protein-free (PF) diet or a regular 25% protein diet (RD). At weekly intervals, lymph node lymphocytes were cultured with PHA in either autologous or pooled 10% rat serum. For weeks 3 through 6, PHA stimulated blastogenesis of lymphocytes from rats maintained on PF diet incubated with autologous serum decreased significantly compared with RD lymphocytes cultured with RD serum. At week 4, PHA blastogenesis of PF lymphocytes cultured with RD serum was similar to that of RD lymphocytes incubated with RD serum. At weeks 5 and 6, PHA stimulation of PF lymphocytes assayed with RD serum was depressed compared with RD lymphocytes cultured with RD serum. For weeks 4 through 6, blastogenesis of RD lymphocytes assayed with PF serum decreased significantly compared with RD lymphocytes incubated with RD serum. Three weeks of protein repletion of rats previously on a PF diet for 6 weeks restored PHA blastogenesis to that observed for lymphocytes from animals in the RD group. The data suggested that suppression of PHA blastogenesis of lymphocytes from rats maintained on PF diet involved a serum factor in the early stages of protein depletion and an additional defect (not serum related) in lymphocyte blastogenesis after prolonged protein depletion. In addition, this defect was corrected after a period of protein repletion.

Lymphocyte responses to mitogen stimulation were studied in healthy hemophiliac patients. To study kinetics and dose response, three different concentrations of the mitogens phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were used, and proliferation was measured on each of 6 days. Patients with T4/T8 ratios of less than 1.00 showed decreased proliferation compared to normal controls and to hemophiliac patients with ratios greater than 1.00. These findings were more pronounced with PWM than with PHA. Over the range of concentrations tested, patients and controls showed similar dose responses to PHA; no dose responses to PWM were demonstrable. There were no significant differences among the patient and control groups in kinetics of response to either mitogen. Interleukin-II (IL-2) activity produced in response to PHA stimulation also was studied. Hemophiliac patients with T4/T8 ratios less than 1.00 showed decreased IL-2 activity in 48-hour PHA culture supernatants compared to normal controls and hemophiliac patients with ratios greater than 1.00.

Nineteen children with iron deficiency, free from factors likely to affect cell mediated immunity (CMI) except four marasmic children and ten age and sexmatched normal controls were studied for their lymphocytic response to PHA stimulation. Lymphoblastoid transformation of patients and controls was comparable. However, four marasmic subjects had lymphoblastoid transformation rates significantly lower to controls. It was, thus,concluded that iron deficiency per se does not affect cellular immunity but concomitant severe malnutrition may be responsible for a depressed response.

In 32 healthy blood donors, 20 patients with histologically verified cancer and 18 patients with non-neoplastic diseases, the fluorescence polarization changes of fluorescein samples incorporated in mononuclear leucotyes were measured after incubation with PHA. The leucocytes of healthy persons, and 16/18 persons with non-neoplastic diseases, responded with a decrease in the degree of fluorescence polarization by about 20% from that in non-PHA-stimulated cells. In 19/20 patients with a variety of malignant tumours, the leucotyes did not respond to PHA stimulation with such a decrease. The exceptions among the patients with neoplastic and non-neoplastic diseases are considered, and may not be "false-negative" or "false-positive" respectively, but indicative of a particular situation in that disease. The biophysical mechanisms underlying the observed changes remain to be investigated. Images Fig. 1

Spontaneous and PHA induced RNA and DNA synthesis was measured qualitatively by autoradiography and quantitatively by scintillation counting in blood lymphoic cells of newborn infants, older infants, and adults. Spontaneous RNA synthesis was found in transitional cells, large phagocytic lymphoid cells and lymphocytes. Transitional cells and phagocytic lymphoid cells also synthesized DNA spontaneously. Quantitatively, spontaneous RNA synthesis was active in both newborn infants and in adults, but significantly less so in older infants. PHA stimulation for 18 h increased RNA synthesis significantly in blood lymphoid cells of newborn infants and adults, and to a much lesser but still significant degree in older infants. Spontaneous DNA synthesis was significantly greater in newborn infants than in older infants and adults. PHA stimulation for 18 h had no effect on thymidine incorporation in any of the groups studied.

Venous blood from eight healthy subjects was divided into four fractions on a discontinuous albumin density gradient. The percentage recovery of lymphocytes was 82.3%; the purity of the lymphocyte fractions was 83.6%. The lymphocytes were cultured with PHA and Endotoxin, and the samples were analysed after 24, 48, and 72 hours. After PHA stimulation immunoblasts appeared up to 59.3% in the cultures from the 19-21% albumin fraction. After Endotoxin stimulation the maximum (75.8%) was reached in the heavy (25-27% albumin) fraction. Thus, it is concluded that the lymphocytes which can be stimulated with both the mitogens have different densities, the PHA-stimulable T lymphocytes being ligther than the Endotoxin-stimulable B lymphocytes. It is also concluded that as a mitogen Endotoxin is equal to PHA.

SummaryLevamisole has been shown to augment the in vitro responsiveness of lymphocytes to PHA stimulation. The response of lymphocytes of 14 (42%) of 33 patients with pulmonary tuberculosis was augmented by levamisole. Augmentation was observed in 9 (64%) of 14 PHA hyporesponsives lymphocytes and in 5 (26%) of 19 normo-responsive lymphocytes.

The bromodeoxyuridine-Giemsa technique has been used to study systematically the incidence of cells in first or subsequent mitoses at different fixation times of human lymphocyte control cultures as well as the influence of ionizing radiations on cell kinetics. Second divisions appear (3%) in cultures harvested 48 h after initiation. In 72 h cultures 40% of the dividing cells are in second and 33% in third division. Administration of 200 rads of X-rays before PHA stimulation results in a mitotic delay but does not increase the incidence of SCE. The yield of dicentrics after an exposure to 200 rads was the same for all cells in first mitosis regardless of fixation time. These results demonstrate that there is no evidence for the existence of sensitive subpopulations that could be distinguished by the time of the first mitotic division following stimulation.

Streptozotocin-diabetic isogenous Brown-Norway (BN) rats received heterotopic Wistar-Furth (WF X BN) F1 heart transplants. The functional graft survival time after different duration of diabetes and during insulin-treated diabetes was recorded. Some diabetic rats were challenged with sheep red cells, and their spleens were used in PHA stimulation and plaque-forming assays. Prolongation in heart transplant survival was found to be independent of the duration of diabetes. The prolongation disappeared promptly when insulin was administered to the diabetic rats. A depression of cellular immunoresponsiveness as measured by PHA stimulation assay was recorded during diabetes. Valid results were not obtained in the plaque-forming assay.

Ten children with selective IgA deficiency were evaluated for cell-mediated immunity (CMI) which included a micromethod for phytohemagglutinin (PHA) stimulation. Standard tests for CMI including the macromethod (400,000 cells) for PHA stimulation were all normal in these patients. However, with the micromethod (2500 cells), the response to PHA of lymphocytes from patients with selective IgA deficiency was significantly depressed ( P = 0.03). Limiting dilution was performed by culturing 400 to 2500 cells with PHA. In five patients there was a significant decrease in the absolute response to PHA at each cell number ( P = 0.02). In this group there was also a diminished slope of the linear regression curves, suggesting that in some patients with selective IgA deficiency the defect in CMI may result from a decrease in the efficiency of T lymphocytes to respond to PHA. To evaluate the mechanism for the increased sensitivity of the micromethod, we did cell counting experiments. We found that while the macromethod resulted in no increase in cell number on the day of peak DNA synthesis, there was a six- to seven-fold increase in cell number in the microcultures indicating that the cell response to PHA in the microcultures is to undergo multiple cell divisions. The enhanced sensitivity of the micromethod may be due to: (i) magnification of a defect in any single cell by 2 N when N is the number of generations that cell will make in culture; and (ii) the diluting out of cell populations which may be limiting to the overall response.

肝疾患患者末梢血のリンパ球をT-cell, B-cellに分け,その比率とそれぞれの機能について検討し,併せて血中HB-Agの検出度と生体の細胞性免疫能の関係についても検討を加えた.さらに肝疾患患者血清中に存在すると考えられるPHA刺激抑制因子についても検索した.すなわち急性肝炎では,極期には細胞性免疫の低下を示し,回復とともに正常に復する.慢性肝疾患では約半数の症例にT-cell populationの減少を,また約40%にPHA反応の低下を認めた.とくにHB-Ag陽性群においては,T-cell population, PHA反応の低下が高率に認められた.各種肝疾患患者19例中8例において,患者血清中に健常人リンパ球のPHA刺激に対する反応を抑制する因子の存在を認めた.これら抑制因子陽性例においては,陰性例に比し有意の差でT-cellpopulationの低下が認められた.以上の成績より肝疾患における細胞性免疫の解析にはリンパ球膜のヒツジ赤血球やPHAに対するreceptorの問題をも含め検討する必要がある.

The presentation of alloantigen to the host via the anterior chamber of the eye can alter the host's immune response. Spleen cells from Lewis rats bearing Brown Norway skin implants in the anterior chamber demonstrated a mildly depressed in vitro blastogenic response to MLC and to PHA stimulation 7-28 days after implantation, a normal response by 35 days, and a slightly greater than normal response by 42 days after implantation. In addition, serum from rats bearing implants for 7-24 days demonstrated a significant depressant effect on the blastogenic response of normal spleen cells to MLC and to PHA stimulation, but the "blocking" effect was absent by 42 days postimplantation. These results indicate that antigen presentation via the anterior chamber of the eye can produce a state of active immunosuppression within the host which could contribute to the privileged nature of the anterior chamber of the eye.

Prior experience with cultures of cerebrospinal fluid lymphocytes indicated a need to develop methods for culturing small numbers of cells. Peripheral blood lymphocytes (PBL) were obtained from 20 normal volunteers. Standard microcultures using 100 X 10(3) PBL/0.2 ml and cultures with 50, 25 and 12.5 X 10(3) in 0.1 ml or 0.2 ml were established in RPMI 1640 with autologous plasma. These cultures were incubated with PHA (1--30 microgram) for 3, 4 and 5 days, pulsed with [3H]thymidine and harvested. In unstimulated cultures, cpm declined linearly with decreasing cell numbers. Standard cultures (100 X 10(3) PBL/0.2 ml) had maximal PHA stimulation (80,916 +/- 6394) at day 3 with 30 microgram PHA. Other 0.2 ml cultures had lower cpm. By culturing 25 X 10(3) PBL in 0.1 ml for 3 days cpm were 82,874 +/- 6875 with 30 microgram PHA and 77,153 +/- 6022 with 15 microgram PHA and were similar to standard cultures. Similar cpm were seen with 12.5 X 10(3) PBL in 0.1 ml after 4 days with 30 micrograms of PHA (80,838 +/- 6674) and with 15 micrograms of PHA (72,860 +/- 6243), and also after 5 days with 30 micrograms of PHA (86,703 +/- 6732) and with 15 micrograms of PHA (74,066 +/- 6388). The maximal response (126,578 +/- 6580) was seen with 25 X 10(3) PBL/0.1 ml at day 4 with 30 micrograms of PHA. By decreasing culture volume to 0.1 ml and increasing time, the number of cells necessary to give PHA responses similar to standard cultures can be reduced by 75--88%.

Immunological function was investigated in patients with acute myeloblastic leukaemia, both in the untreated stage of the disease and in remission. IgM concentrations were found to be raised in 7 out of 29 patients during the untreated stage. There were only minimal changes in 1gG, 1gA and C' concentrations, and in the incidence of auto-antibodies to normal tissue components. Reactions to standard skin tests were considerably impaired--only 2 out of 10 leukaemic patients in remission responded to 2 or more of these tests. Furthermore the response in leukaemic patients was much weaker than in the corresponding controls. PHA stimulation of lymphocytes from patients in remission showed considerable variation from near normal to gross impairment but a response below 40% of normal was associated with a short remission period, suggesting that PHA stimulation may be a useful indication of the likelihood of relapse.

Wasrige Extrakte aus Kalbsmilz wurden durch Azetonfallung, Membranfiltration, Affinitatschromatographie und Dialyse gereinigt. Diese Extrakte hemmten den Thymidineinbau in lymphoiden Zellen (MKT-CH und PHA-stimulierte Lymphocytenkulturen). Zellen nicht lymphatischen Gewebes (Melanomzellen Mel Ei 78 und Ehrlich-Ascites-Zellen) wurden nicht beeinflust. Die Hemmung war reversibel, und die Reinextrakte wirkten nicht zytotoxisch. Die Extrakte entsprechen einem Chalon. Ihre mogliche Bedeutung fur die Verhinderung der Graft versus host reaction und fur die Behandlung lymphoproliferativer Erkrankungen wird diskutiert.